The research in Dr. Elkins' laboratory concerns the fundamental mechanisms of protective immunity to intracellular pathogens, particularly intracellular bacteria. Current projects focus on role of lymphocytes, natural killer cells, neutrophils, macrophages, and their products (both antibodies and cytokines), in short and long term immunity to Mycobacterium tuberculosis and Francisella tularensis LVS (a model gram negative intracellular bacterium). Studies are directed at understanding the transition from innate to specific immunity, the mechanisms of T cell mediated control of intracellular bacterial growth (especially effector mechanisms other than interferon gamma), early events in lung granuloma formation following M. tuberculosis infection, the contribution of Toll-like receptors to initiation of resistance to Francsella infection, and the role of B cells in intracellular infections. Companion studies address the role of recognition of bacterial DNA during infection in innate immunity. We are particularly interested in the similarities and differences between the host immune responses following systemic or aerosol infection with both bacteria. Collaborative projects include the study of host genes that control susceptibility to intracellular pathogens; the role of chemokines in responses to intracellular infections; interactions between B cells and natural killer cells; identification of virulence factors of Francisella, and genes required for growth in macrophages; bacterial DNA as a therapy for M. tuberculosis infection; and development of DNA vaccines against herpes simplex virus. This research program is concerned with elucidation of the basic mechanisms of protective immunity to intracellular bacterial pathogens, including Mycobacterium tuberculosis, Francisella tularensis, and Listeria monocytogenes. A better understanding of the nature of protective immunity is essential to the rational design of new or improved vaccines, and prediction of useful correlates of protection. Thus we are characterizing the pathology of infection, the cell types involved, their cell-cell interactions and products, the specificity of the responses, the recognition receptors used, and the nature of the cellular responses provoked in the context of in vivo primary and secondary intracellular bacterial infection. Murine infection with Francisella tularensis Live Vaccine Strain (LVS), a gram negative facultative intracellular bacterium that replicates in macrophages, allows concurrent study of sublethal infection, lethal infection, and immune memory. Protective immune responses to F. tularensis also appear similar to those of M. tuberculosis (M. tb., TB) and L. monocytogenes. Thus each can be studied as representative of this class of pathogens, with a view toward identifying common or distinct patters of immune responses to intracellular bacteria. Because LVS is the only current vaccine candidate for tularemia in the United States, it is also of clinical interest to understand mammalian immune responses to this bacterium. Studies this year have focused on several areas, including: the roles of T cells, B cells, interferon gamma and chemokine receptors in resolution of primary and secondary LVS and M. tb. infection; early events, including the role of B cells, in establishment of lung pathology and dissemination of M. tuberculosis; and the role of recognition of bacterial DNA containing CpG motifs by the innate immune system in host response to LVS and M. tuberculosis. Most of these studies took advantage of a newly developed, novel in vitro culture system that replicates many of the features of in vivo infection. Bone marrow macrophages infected with LVS or M. tb. supported exponential growth of bacteria; addition of spleen or lung cells from LVS-immune or TB-immune mice specifically controlled LVS or M. tb. intracellular bacterial growth (respectively). This culture system therefore permits direct study of control of intracellular bacterial growth without any assumptions about the mechanisms of control. Using infected macrophages from interferon gamma receptor KO mice, we have demonstrated that there are significant mechanisms of immunity for both pathogens that are independent of signalling through macrophages' interferon gamma receptors. These included contact-dependent mechanisms such as FasL, but not perforin, and other mechanisms such as elaboration of TNF alpha. In studies of early events in development of pathology and dissemination of M. tb., we are using transgenic mice that contain mature B cells but lack M cells as well as the ability to secrete antibodies to examine the relative contributions of each to M. tb. lung pathology. To date these studies suggest that lung B cells, but not specific antibodies, control development of lung pathology and dissemination. Studies this year have further demonstrated that survival of primary sublethal LVS infection is critically dependent on MyD88, a major adaptor molecule in the signal transduction pathway for Toll-like receptors, but not on TLRs 2, 4, or 9. Further, innate immune responses to LVS depend on LRG-47, a GTPase that is a member of a newly described family of interferon-inducible proteins. In studies on protection provided by DNA containing CpG motifs, we demonstrated that control of intracellular bacterial growth by DNA-primed lymphocytes is dependent entirely on soluble mediators, including interferon gamma, IL12, and TNF alpha. While natural killer cells are activated by CpG DNA and produce interferon gamma, NK cells are not absolutely required for in vivo protection. Further understanding of such immunobiological properties of bacterial DNA will be important in evaluation of DNA vaccines, the use of bacterial DNA as an adjuvant, and therapeutic applications of bacterial DNA. Because LVS is a live attenuated strain, better methods to assess potency and consistency of manufacture of this vaccine candidate are of interest. We have therefore compared several lots of LVS in terms of growth in macrophages, murine virulence, and a panel of molecular markers. Although no obvious differences were found at the molecular level, subtle but consistent differences in virulence in two strains of inbred mice have been found. We are currently developing flow cytometry based techniques for quantitating live vs. dead bacteria in production lots and simultaneously evaluating the extent of LPS phase variation in those lots.